Many important pathogens circulate within multi-host communities 2. Equally, many hosts can be infected with multiple pathogens at any given time 3. Recent studies of declining amphibian populations indicate that the two pathogens of most concern, Ranavirus and Batrachochytrium dendrobatidis (Bd), commonly occur as co-infections 4. Yet, experimental evidence of co-infection dynamics in amphibians remains sparse.
While both pathogens are host generalist, significant heterogeneity exists in host susceptibility, infectiousness and burden of each disease, at a species and individual level 5. Which conforms with a wider body of literature showing host species unevenly contribute to disease transmission in multi-host communities 6. As a result, pathogen persistence at a community level is largely dictated by the composition of host species 7. Predicting how these heterogeneities in disease contribution across different host species relates to the establishment and persistence of a pathogen in a host community, revolves around understanding the transmission process between the infectious stage of a pathogen and a susceptible individual 8.
To help unpick the tangled dynamics of this multi-host, multi-pathogen system we conducted an experiment looking at how susceptibility, infectiousness and burden of disease changed with infection scenario (single vs coinfection) across a panel of host species (Bufo bufo, Rana temporaria and Alytes muletensis) that range in their reported susceptibility. We measured the contributions of each host, at an individual level, to the environmental pool of infectious particles, by quantifying Bd zoospores and ranavirus virion outputs for four days post exposure. Endpoint infection load of each host was also measured to link the hosts’ infection burdens with their infectiousness.
Understanding the susceptibility and infectiousness of each host at an individual level allows us to predict how host species community composition influences the establishment and persistence of both pathogens, singularly and as coinfections, at the community level. Our study provides previously lacking emperical evidence of within-host and between-host dynamics under different infection scenarios (single vs co-infection) and highlights the importance of understanding host contributions to enviromental reservoirs of disease.
I. Ascertain whether the sequence of exposure to co-infecting pathogens (Bd and Rv) alters the disease outcome for the host and subsequent transmission of the pathogens.
II. Establish the contributions of hosts to the environmental “pool” of infectious particles, across species at an individual level, to understand variations in host infectiousness and by proxy transmission.
In order to capture a range of host susceptibilities to infection by Bd and/or Rv I examined shedding rates and infection intensity of two co-occurring species (B. bufo & R. temporaria) that display contrasting resistances, and one other species (A. muletensis) documented to respond distinctly to Bd exposure but which has no empirical records of ranavirus exposure.
B. bufo and R. temporaria clutches were collected, with the permission of the landowner and under licence, from a pond site in Henley as spawn and hatched out in an approved facility. Larval A. muletensis were reared from egg clutches by the ZSL Living Collections.
The total number of animals that underwent procedures (includes controls that had sham bath exposures).
Bufo bufo = 220, Rana temporaria = 110, Alytes muletensis = 107
Experiments were run in batches comprising of two B.bufo experiments of 100 animals, with 10 reference animals, a R.temporaria experiment, also with 10 reference animals, one experiment of 70 A.muletensis tadpoles, with all treatment groups, and one Bd-only experiment with less developed A.muletensis (n=37). Each treatment group had n=20 bar the A.muletensis. The larger A.muletensis (here on in referred to as A.muletensis I) had n=14 per treatment and the smaller A.muletensis (A.muletensis II) had n=25 in the Bd-only exposure treatment group and n=12 tadpoles as controls.
The number of tadpoles used in the experiments is based on previous experiments where the lab group have investigated such parameters as endpoint infectious burdens.
Reference animals were euthanised on exposure day 12 (see Figure 1) as a means of assessing whether the exposure schedule resulted in infection. Before euthanasia, the reference animals underwent the shedding/filtering process (see relevant method section below) so we could compare early stage shedding rates with endpoint infection loads and to initially trial the procedure on live samples.
Tadpoles were transferred to the experimental rooms and into individual housing once the gills were reabsorbed, free swimming and exhibit normal behaviour. For each species, larvae of similar mass and developmental stage (Gosner 25; Gosner, 1960) were randomly selected, acclimatised to the individual housing (Really Useful Boxes (RUBs)) of the appropriate size for the species (see below) and then randomly assigned to a treatment group. As outlined in Figure 1, treatment groups consisted of 20 individuals per species.
All individuals were checked and rotated daily, left to right between treatments and front to back within treatments.
| Experiment | RUB size | exposure water vol. | housing water vol. |
|---|---|---|---|
| Bufo bufo I | 0.075L | 210mL | 375mL |
| Bufo bufo II | 0.075L | 210mL | 375mL |
| Rana temporaria | 0.075L | 210mL | 375mL |
| Alytes muletensis I | 0.7L | 520mL | 1000mL |
| Alytes muletensis II | 0.075L | 210mL | 375mL |
Larval amphibians were fed 200µL of ground Tetra Tabimin tablets dispersed in double-distilled water with a ratio of 1g/100mL, every other day during the experiment. Tadpoles in the first Bufo experiment were fed 300µL for the first few feeds but it was evident they were not consuming all the food so it was reduced down to 220µL. The older A.multenesis (II) tadpoles recieved double the feed (~400µL) due to their larger size.
All the Alytes muletensis received a slightly altered diet. Half of the ground Tetra Tabimin was subsituted with coarse ground Spirulina tablets, in an attempt to improve water quality.
In hindsight I think the volume of food was still too high but unfortunately we didn't record tadpoles weight change so we have no emperical evidence.
Full water changes (100%) were made every 4 days throughout the course of the experiment or before an exposure. A 15ml water samples was collected before the 100% water change pre-exposure and from the hosuing and shedding container after the shedding period for four sentinel tadpoles per experiment.
Water quality was an issue throughout the experimental period. Steps have been taken to resolve this issue for future experiments mainly by the installation of Reverse Osmosis units in the animal rooms - thanks to Chris S.
The temperature of the animal rooms followed natural ambient conditions (18-24oC) and was monitored throughout. The summer heatwave alongside faulty temperate control units meant temperature fluctuated significantly. See Table 2 for minimum and maximum recordings during each experiment. Free standing air-con units were installed as a temporary, emergency measure to control room temperatures by the begining of August 2018. Meaning the temperature for the A.muletensis experiments was carefully controlled.
| Experiment | min. temp. (oC) | max. temp. (oC) |
|---|---|---|
| Bufo bufo I | 16.6 | 23.5 |
| Bufo bufo II | 16.7 | 27.6 |
| Rana temporaria | 16.7 | 27.6 |
| Alytes muletensis I & II | 15 | 16.6 |
Table 1. Minimum and maximum temperatures during each experiment measured by thermometer placed in the middle of the animal room. The temperatures for the A.muletensis experiments are readings from a thermometer in the water vat.
Temperature control was an problem for the majority of the experiments. Unfortunately, despite best efforts (thanks again to Chris S.) it looks to be an ongoing issue.
Tadpoles were exposed individually to controlled doses of Bd and/or Rv or the correlating sham media (see Figure 1). The exposure inoculum was added directly to the housing container after a 100% water change. Following the 6-hour exposure period the water volume in the housing containers was raised to maintain water quality.
Figure 1: Schedule of exposures and sampling, by treatment group. A dose is denoted by a green (Rv) or yellow (Bd) coloured square, and sham doses are displayed as cross-hatched squares. The blue squares indicate when 50ml “soak” water samples were collected from individuals and filtered. EMA water samples (for quantification of viable Bd zoospores) are collected directly from the housing container and shown by an asterisk.
Ranavirus strain: isolate RUK13 9 was cultivated on epithelioma papillosum carp (EPC) cell lines at 18oC and 5% CO2 (courtesy of S.J.Price, C.Owen and L. Brookes), and quantified using the TCID50 method 10. The harvested cell culture fluid contained a virus titre of 107 TCID50/ml.
Bd strain: IA’9’13, a member of the hypervirulent BdGPL lineage isolated during an epidemic at Ibón Acherito (Pyrenees, Spain) in 2013 by Prof. M. Fisher, was cultured in TGhL broth, in a 25cm2 cell culture flasks, at 18oC. Zoospores were collected and counted using a haemocytometer.
Ranavirus Designated individuals were exposed to 105µL (or 263µL for A.muletensis II) meaning an effective exposure of 104.5 TCID50/ml. The dose was deemed suitable based on previous work where similar inoculums induced infection but had a longer time till death in tadpoles then higher concentrations 11.
Bd A Bd positive dose consistent of 15,000 to 600,000 active zoospores in 210µL liquid media, or 525µL for A.muletensis II. The volume of media was standardised across doses in order to maintain water quality during the exposure period. Total exposure are shown in the table below.
| Experiment | total zoospores |
|---|---|
| Bufo bufo I | 3,675,000 |
| Bufo bufo II | 1,443,750 |
| Rana temporaria | 2,336,250 |
| Alytes muletensis I | 472,500 |
| Alytes muletensis II | 294759.9 |
At the end of the experiment (day 9 post-exposure) tadpoles were euthanised, under licence, by buffered (pH 7.0) 5 mg/L tricaine methylsulfonate (MS-222) and then stored in 100% ethanol.
The Bd infection load of tadpoles was confirmed by excision and DNA extraction of mouthparts, the site of Bd colonization and infection in tadpoles, using Prepman Ultra (Life Technologies) as per Hyatt et al. (2007). Extracts were screened by the qPCR diagnostic outlined in Boyle et al. (2004) which targets the ITS-1 and 5.8S regions and diluted 1:10 before qPCR to avoid inhibition. The infection load with be express in genomic equivalents (GE), where one GE is equivalent to a single zoospore.
The quantification of ranaviral DNA from tissue samples (mainly livers and kidneys) of the tadpoles stored in 100% ethanol was by DNeasy Blood and Tissue (Qiagen) extraction following the manufacturers protocol. DNA samples were then analysed with a qPCR assay specific to the ranaviral major capsid protein (MCP) sequence and normalised by host cell quantity as outlined in Leung et al. (2017).
Figure 2: Left DNA extraction procedure for endpoint tissue samples. Right
One limitation of using common frog and common toad larvae is that individuals can not be sampled for infections without first being euthanized. For this reason, we tracked infectious particle output post-exposure using a modified version of the “soak” technique 12. Individuals were transferred into a temporary shedding unit containing clean, aged water to “soak” for 4 hours, after which they will be returned to their housing unit. The shedding containers used are shown in Figure 3. The soak period of 4 hours was choosen to balance the need to sensitively detect zoospores/virions shed with the considerations of the tadpoles welfare needs.